陳志林，馮美瑩，陳預(yù)明，衛(wèi)恒習(xí)，李莉，吳同山，張守全

1. 華南農(nóng)業(yè)大學(xué)動(dòng)物科學(xué)學(xué)院，國(guó)家生豬種業(yè)工程技術(shù)研究中心，廣州 510642；

2. 東莞市畜牧科學(xué)研究所，東莞 523086

哺乳類動(dòng)物受精是一個(gè)精細(xì)的程序化復(fù)雜過(guò)程。受精過(guò)程中任何的偏差往往會(huì)導(dǎo)致動(dòng)物的低繁殖力或不育[1]。具有良好受精能力的精子是受精成功的先決條件。精子在睪丸曲精細(xì)管中不斷生成，隨后離開睪丸進(jìn)入到附睪中進(jìn)行成熟。在這一過(guò)程中，精子發(fā)生了若干的生理變化，其主要表現(xiàn)為附睪及副性腺分泌的蛋白因子在精子頭部的組裝和附著，精子質(zhì)膜表面蛋白構(gòu)象的改變，向前運(yùn)動(dòng)能力的獲得等[2]。精子在雌性生殖道里需要依次經(jīng)歷超活化和獲能反應(yīng)，以獲得與卵子受精的能力。獲能的精子需要依次通過(guò)子宮與輸卵管接合部、輸卵管壺-峽連接部等屏障才可以與卵子相遇。只有運(yùn)動(dòng)活力極強(qiáng)的精子才能夠通過(guò)雌性生殖道中的這一系列屏障。在精卵相遇后，精子仍需要經(jīng)歷復(fù)雜的過(guò)程才能完成受精過(guò)程。其中，精卵開始接觸的過(guò)程中，精子受卵丘細(xì)胞和透明帶的作用，逐步誘發(fā)了頂體反應(yīng)；反應(yīng)過(guò)程釋放多種頂體內(nèi)的水解酶以促進(jìn)精子溶解透明帶；同時(shí)，頂體膜與精子質(zhì)膜的融合暴露出精子頭部的卵識(shí)別和結(jié)合位點(diǎn)[3]，為隨后的精卵結(jié)合做好準(zhǔn)備；精子溶解透明帶后進(jìn)入到卵周間隙，精子頭部的識(shí)別和結(jié)合位點(diǎn)在若干蛋白因子的協(xié)助下，識(shí)別和結(jié)合卵膜受體蛋白[4]，從而導(dǎo)致精卵的膜融合(圖1)，并最終形成完整的受精卵。

圖1 受精過(guò)程

在整個(gè)受精過(guò)程中，精子功能相關(guān)的蛋白分子參與其中的信號(hào)通路調(diào)控以及一系列的生理生化反應(yīng)。隨著蛋白組學(xué)的發(fā)展和基因敲除技術(shù)的應(yīng)用，哺乳動(dòng)物精子功能相關(guān)的蛋白質(zhì)不斷涌現(xiàn)。本文主要對(duì)精子相關(guān)的信號(hào)通路及其他受精過(guò)程相關(guān)的精子功能蛋白質(zhì)進(jìn)行總結(jié)，闡述這些蛋白質(zhì)分子與精子運(yùn)動(dòng)活力、精子獲能、頂體反應(yīng)、透明帶穿入以及精卵融合方面的關(guān)系，并對(duì)其在改善哺乳動(dòng)物繁殖力上的應(yīng)用前景提出展望。

1 精子運(yùn)動(dòng)活力及獲能反應(yīng)的信號(hào)通路

在受精過(guò)程中，精子不僅需要在雌性生殖道中游動(dòng)到與卵子結(jié)合的位點(diǎn)，而且還要獲得與卵子受精的能力，故精子運(yùn)動(dòng)活力及獲能反應(yīng)是影響受精的兩個(gè)關(guān)鍵因素。精子運(yùn)動(dòng)活力主要由精子尾部的長(zhǎng)鞭毛擺動(dòng)而形成[5]，除了受外界環(huán)境影響外，也受到內(nèi)部若干種信號(hào)通路的調(diào)節(jié)。精子獲能是精子獲得與卵子結(jié)合的能力的生理過(guò)程，是精子在受精前必須經(jīng)歷的一個(gè)重要反應(yīng)。環(huán)腺苷酸(Cyclic adenodine monophosphate, cAMP)-環(huán)腺苷酸依賴性蛋白激酶A(CAMP-dependent protein kinase, cAMPPKA)信號(hào)通路和鈣離子信號(hào)通路是調(diào)控哺乳動(dòng)物精子運(yùn)動(dòng)活力和獲能反應(yīng)的兩個(gè)最重要的信號(hào)通路，其信號(hào)通路調(diào)控模式相對(duì)比較清晰[6](圖2)。這兩種信號(hào)通路的活化過(guò)程分別是由精子細(xì)胞內(nèi)的 HCO3-和Ca2+水平來(lái)調(diào)節(jié)[7]。自精子從雄性生殖道射出后，精清富含的HCO3-可以通過(guò)Na+/ HCO3-協(xié)同轉(zhuǎn)運(yùn)蛋白(Cotransporter, nbc)進(jìn)入精子細(xì)胞內(nèi)，引起胞內(nèi)的pH改變[8]。與此同時(shí)，由精子膜產(chǎn)生的膽固醇外流作用促使膜通透性的增強(qiáng)，HCO3-也因此可以快速進(jìn)入精子細(xì)胞內(nèi)[9]，引起細(xì)胞內(nèi) HCO3-濃度的升高，導(dǎo)致精子胞質(zhì)的堿性化。胞質(zhì)堿性化在刺激精子加速新陳代謝的同時(shí)激活了胞內(nèi)的可溶性腺苷酸環(huán)化酶(Soluble adenyl cyclase, sAC)[10]。sAC的活化過(guò)程是增強(qiáng)精子運(yùn)動(dòng)活力和誘發(fā)精子獲能的必要過(guò)程。sAC的主要作用是引起細(xì)胞內(nèi)cAMP水平的提高，并致使 PKA的激活[6]?；罨?PKA可以使精子細(xì)胞內(nèi)的蛋白磷酸酶(Protein phosphatases，PPs)活性受到抑制，同時(shí)也不斷地激活蛋白酪氨酸激酶(Protein tyrosine kinases, PTK)，使胞內(nèi)蛋白酪氨酸磷酸化作用得到增強(qiáng)。另外，精子內(nèi)PKA的活化還有利于鈣離子通道蛋白(CatSper)的激活，從而達(dá)到誘發(fā)精清中的Ca2+內(nèi)流的效果[11～13]。胞內(nèi)Ca2+濃度的升高不僅可以引起精子細(xì)胞膜的超極化[14]，增強(qiáng)精子鞭毛的運(yùn)動(dòng)活力[15,16]，也能夠維持sAC的活化程度[6]，從而持續(xù)激活 PTK，增強(qiáng)胞內(nèi)蛋白酪氨酸磷酸化的水平。事實(shí)上，PTK的活化能夠促使凝溶膠蛋白(Gelsolin)發(fā)生磷酸化[17]，并使其處于抑制狀態(tài)，進(jìn)而引起肌動(dòng)蛋白的聚合作用，最后引發(fā)精子發(fā)生超活化反應(yīng)[18,19]。另外，由于PPs去磷酸化活性被抑制以及PTK磷酸化作用的持續(xù)，細(xì)胞內(nèi)的蛋白酪氨酸磷酸化水平不斷提高，從而誘發(fā)精子超活化運(yùn)動(dòng)和獲能反應(yīng)[20,21]。精子的超活化和獲能不僅直接活化了精子鞭毛軸絲[16]，增強(qiáng)鞭毛運(yùn)動(dòng)性和尾部側(cè)擺幅度[15,22]，還解除了對(duì)精子頂體反應(yīng)的抑制，有利于精卵的結(jié)合。研究表明，胞內(nèi) HCO3-和 Ca2+高濃度水平的狀態(tài)不僅是維持精子超活化運(yùn)動(dòng)和獲能反應(yīng)的關(guān)鍵[11,23]，也是精子發(fā)生頂體反應(yīng)的重要因素[6]。

圖2 cAMP/PKA信號(hào)通路和鈣離子信號(hào)通路

2 參與精子運(yùn)動(dòng)活力調(diào)節(jié)及獲能反應(yīng)的精子功能蛋白

雖然cAMP/PKA信號(hào)通路和鈣離子信號(hào)通路的具體分子調(diào)控機(jī)理仍不明確，但近年來(lái)的研究結(jié)果表明，多種精子和精清蛋白質(zhì)參與這些過(guò)程(表 1)。其中鋅 a2糖蛋白(Zn-a2-glycoprotein, ZAG)是調(diào)節(jié)cAMP/PKA通路中的關(guān)鍵因子cAMP的蛋白分子。ZAG抗體能夠顯著地降低精子細(xì)胞內(nèi)的cAMP水平，致使PKA蛋白激酶不能維持活化狀態(tài)，從而抑制精子的超活化，影響精子的運(yùn)動(dòng)活力[24]。附睪活力抑制因子II (Motility inhibiting factor, MIF-II)[25]和精漿活力抑制因子(Seminal plasma motility inhibitor,SPMI)[26]與 ZAG抗體作用相似，同樣具有降低精子細(xì)胞內(nèi)cAMP濃度的功能，但其具體作用靶點(diǎn)仍不清楚。此外，CatSper是鈣離子信號(hào)通路的關(guān)鍵蛋白。活化的 CatSper能夠引起 Ca2+內(nèi)流，誘發(fā)精子發(fā)生超活化反應(yīng)。研究表明，缺失catsper基因的小鼠精子失去超活化的能力以及表現(xiàn)出極弱的運(yùn)動(dòng)力[27]。另外，小鼠附睪分泌的鈣離子-腺苷三磷酸膜蛋白(Plasma membrane Ca2+-ATPase 4a, PMCA4a)能夠作為一種 Ca2+流泵，影響鈣離子信號(hào)通路的激活并維持精子內(nèi)Ca2+的穩(wěn)態(tài)。缺失pmca4基因的精子不能夠發(fā)生超活化和獲能反應(yīng)[28]，因此PMCA4a對(duì)精子運(yùn)動(dòng)活力和受精具有極其重要的作用。

表1 精子運(yùn)動(dòng)活力及獲能相關(guān)的蛋白質(zhì)

公牛精清蛋白(Bovine seminal plasma, BSP)是與公牛繁殖力相關(guān)的蛋白質(zhì)。BSP可以改變精子膜表面的蛋白構(gòu)象，增強(qiáng)精子膜的流動(dòng)性，調(diào)節(jié)膜表面的膽固醇外流，從而參與精子獲能的調(diào)節(jié)[29]。研究表明，BSP的主要成分PDC-109是引起膜膽固醇流的主要作用蛋白[30]。PDC-109能夠改變膜的通透性，提高胞內(nèi)的HCO3-和Ca2+-ATPase的濃度，影響精子的運(yùn)動(dòng)活力以及獲能進(jìn)程[31]。

隨著對(duì)受精生物標(biāo)記的深入研究，多種精子活力及獲能相關(guān)的蛋白質(zhì)也隨之被發(fā)現(xiàn)。致密纖維蛋白(Outer dense fiber protein, ODF2)是精子尾部的組成蛋白，對(duì)精子尾部的擺動(dòng)作用十分重要。研究顯示，odf2+/-嵌合體小鼠由于產(chǎn)生運(yùn)動(dòng)活力極低的精子而重度不育[32]。附睪蛋白酶抑制蛋白(Epididymal protease inhibitor, EPPIN)是一種由睪丸支持細(xì)胞分泌的蛋白，能夠與精囊蛋白相互結(jié)合，抑制精子的活力[33]。Trophinin-binding Peptide是發(fā)現(xiàn)于精子尾部的一種內(nèi)源性蛋白質(zhì)，能夠促進(jìn)精子尾部的擺動(dòng)，增強(qiáng)精子的直線游動(dòng)能力[34]。絲氨酸蛋白酶抑制因子(Serine protease inhibitor kazal-type-like SPINKL)是一種由精囊分泌的蛋白因子[35]，能夠抑制精子膜的膽固醇外流，阻礙膜通透性的增強(qiáng)和胞質(zhì)的堿性化，從而抑制精子發(fā)生獲能反應(yīng)[36]。Sp32作為前頂體素的結(jié)合蛋白，發(fā)生磷酸化作用主要是與精子獲能相關(guān)[37]。表皮生長(zhǎng)因子受體(Epidermal growth factor receptor, EGFR)獲能時(shí)的磷酸化則可能與獲能信號(hào)通路相關(guān)，因?yàn)镋GFR與表皮生長(zhǎng)因子(Epidermal growth factor, EGF)結(jié)合后，不僅能夠明顯地降低精子蛋白質(zhì)酪氨酸的磷酸化作用[38]，且抑制了PKA的活化狀態(tài)[9]。此外，酶檸檬酸蛋白酶、延胡索酸水化酶、蘋果酸酶和脫氫酶在公馬精液中存在，其含量與精子運(yùn)動(dòng)活力和個(gè)體的受精能力呈正相關(guān)[39]；公牛精液的骨橋蛋白(Osteopontin, OPN)對(duì)于維持精子在母性生殖道中的活力及獲能狀態(tài)具有重要作用，其含量與雄性個(gè)體繁殖力呈正相關(guān)[40]。

上述蛋白對(duì)精子運(yùn)動(dòng)活力或獲能都具有重要的作用，但其作用機(jī)理還有待考證。在今后的研究中，通過(guò)這些蛋白篩選高繁殖力的個(gè)體，檢測(cè)這些分子標(biāo)記以判斷是否留種，從而指導(dǎo)畜牧生產(chǎn)育種工作，提高選育工作效率。

3 精子功能蛋白質(zhì)與頂體反應(yīng)的關(guān)系

獲能后的精子在輸卵管中與卵子相遇后，接觸并溶解卵丘細(xì)胞層。在該過(guò)程中，精子運(yùn)動(dòng)活力和透明質(zhì)酸酶的作用十分關(guān)鍵。在自然受精時(shí)精子穿越卵丘層的分子機(jī)理至今仍不清楚，一般認(rèn)為是精子頂體在穿透卵丘細(xì)胞時(shí)能夠誘發(fā)頂體膜的融合、頂體內(nèi)各種酶的釋放以及卵透明帶被水解等頂體反應(yīng)現(xiàn)象(圖3)。然而有研究顯示，在精子成功穿過(guò)卵丘細(xì)胞層后，頂體的配體與透明帶表面的受體相互識(shí)別，膜表面的半乳糖基轉(zhuǎn)移酶(GalT)等蛋白分子隨后與透明帶蛋白(Zona pellucida protein, ZP3)結(jié)合，引發(fā)頂體反應(yīng)[41]。盡管頂體反應(yīng)的發(fā)生仍存在爭(zhēng)議，但反應(yīng)形成的入卵通道以及過(guò)程中暴露出的卵膜識(shí)別位點(diǎn)對(duì)精卵融合過(guò)程必不可少。

圖3 頂體反應(yīng)的過(guò)程

頂體反應(yīng)是受精的先決條件，只有經(jīng)過(guò)獲能和完成頂體反應(yīng)的精子才能夠進(jìn)入卵周間隙，完成精卵膜融合過(guò)程。該反應(yīng)時(shí)間進(jìn)程長(zhǎng)，多種蛋白分子參與其中(表2)。除了GalT能夠與ZP3結(jié)合外，乙酰膽堿受體(Acetylcholine receptor, α7nAChR)能夠致使 EGFR分子活化，介導(dǎo)EGFR和 ZP3結(jié)合[42]。EGFR和ZP3的結(jié)合有可能是刺激了鈣離子通路，影響 Ca2+調(diào)節(jié)的頂體反應(yīng)。另外，肌動(dòng)蛋白結(jié)合蛋白(Secretory actin-binding protein, SABP)是一種位于精子尾部的蛋白分子，在頂體反應(yīng)時(shí)與肌動(dòng)蛋白結(jié)合，從而阻斷肌動(dòng)蛋白的聚合[43]。由于精子獲能后Gelsolin發(fā)生的去磷酸化能使肌動(dòng)蛋白解聚，并引發(fā)頂體反應(yīng)，因此 SABP很可能是一種誘發(fā)頂體反應(yīng)的重要作用因子。此外，精子頭部還存在多種與頂體反應(yīng)相關(guān)但作用位點(diǎn)未知的蛋白。P14是一種附睪來(lái)源的頂體蛋白質(zhì)，在頂體膜與精子質(zhì)膜發(fā)生融合時(shí)發(fā)揮重要的作用，其抗體能夠顯著降低頂體反應(yīng)的發(fā)生率[44]。與P14作用相似的還有動(dòng)力蛋白(Dynamin)，Dynamin能夠啟動(dòng)頂體內(nèi)容物胞外分泌，調(diào)控頂體膜融合的發(fā)生[45]。此外，精子頭部的頂體相關(guān)蛋白分子(Sperm acrosome associated 7, SPACA7)的釋放也是誘發(fā)頂體反應(yīng)的關(guān)鍵因素[46,47]。

表2 精子頂體反應(yīng)相關(guān)的蛋白質(zhì)

4 精子功能蛋白質(zhì)與入卵通道的形成

哺乳動(dòng)物卵透明帶一般由 3～4種糖蛋白(ZP1、ZP2和ZP3等)組成[49,50]，構(gòu)成了精卵受精過(guò)程的屏障。自發(fā)生頂體反應(yīng)后，頂體內(nèi)釋放的復(fù)合酶(頂體素(Acrosin)、卵結(jié)合蛋白、脂酶和唾液酸苷酶等)能溶解卵透明帶，協(xié)助精子突破此屏障，并形成一條入卵通道。事實(shí)上，在形成入卵通道之前，精子頭部需要結(jié)合在透明帶表面以形成一種牢固的狀態(tài)(圖4)。這種狀態(tài)對(duì)于精子隨后溶解堅(jiān)硬的透明帶是十分必要的。精子頭部?jī)?nèi)源性或附著的蛋白則是形成該狀態(tài)的關(guān)鍵分子(表3)。

圖4 精子穿入透明帶的過(guò)程

表3 精子穿入透明帶相關(guān)的蛋白質(zhì)

近年來(lái)相關(guān)的研究結(jié)果還發(fā)現(xiàn)了與穿入透明帶相關(guān)的 Acrosin[51]、RNase10[52]、Prss37[53]和 PMIS2[54]4種蛋白質(zhì)。這 4種蛋白質(zhì)在精子與透明帶形成穩(wěn)定的綁定或結(jié)合狀態(tài)時(shí)發(fā)揮著必不可少的作用。敲除編碼這 4種蛋白的任一基因后，小鼠精子均不能與卵子的透明帶產(chǎn)生相互作用，也無(wú)法完成兩者間的綁定與結(jié)合，從而導(dǎo)致受精失敗。哺乳動(dòng)物附睪合成的精子粘附素(Heparin-binding spermadhesins,AQN-3)[55]和 Lactadherin[48]同樣能夠協(xié)助精子與透明帶發(fā)生結(jié)合，使精子可以牢固地綁定在卵子表面。精細(xì)胞內(nèi)源的Testase1蛋白因子則能夠發(fā)生磷酸化，并作為一種錨定蛋白來(lái)引導(dǎo)頂體復(fù)合酶溶解透明帶，實(shí)現(xiàn)入卵通道的形成[56]。雖然這些功能蛋白質(zhì)的具體作用方式不明確，但目前多數(shù)推斷是與透明帶表面受體相互結(jié)合，保證精子穩(wěn)定地結(jié)合在卵子上，為溶解透明帶提供一種穩(wěn)態(tài)，也為精子順利穿入卵子做好鋪墊。

5 精子功能蛋白質(zhì)與精卵融合過(guò)程的關(guān)系

精子穿過(guò)透明帶后進(jìn)入到卵周間隙。此時(shí)，精卵膜融合的成功是精子頭部?jī)?nèi)的遺傳物質(zhì)輸送入卵細(xì)胞內(nèi)的保證，也是受精過(guò)程最重要的一步。雖然精卵融合的具體過(guò)程和分子機(jī)制目前尚不十分清楚，但是精子蛋白質(zhì)在這個(gè)過(guò)程中發(fā)揮著不可替代的作用(表4)。其中熱休克蛋白(Heat shock 70 kDa protein 2, HSPA2)與精子頭部粘附因子(Sperm adhesion molecule 1, SPAM1)形成識(shí)別復(fù)合物，介導(dǎo)精卵間的識(shí)別[57]；隨后 Phospholipase C-zeta[58]和 Prosaposin[59]能夠激活卵子，刺激精卵發(fā)生膜融合(圖5)，實(shí)現(xiàn)合子的形成。精卵膜融合的實(shí)質(zhì)是精子膜和卵膜表面互補(bǔ)配對(duì)的特異性蛋白分子介導(dǎo)的過(guò)程，如精子膜表面的受精素家族(ADAMs)和卵膜上的整合素(Integrin)配對(duì)[60,61]，精子頭部赤道區(qū)的IZUMO1蛋白和卵膜上JUNO分子的結(jié)合[62～64]，精子頂體后膜的 CRISP家族和卵膜上微絲的相互作用[65,66]，IAM38[67]和Zonadhedin[48]分別與不同卵膜受體蛋白相互結(jié)合等。另外，卵膜上CD9分子、微絲的長(zhǎng)度及密度對(duì)精卵膜識(shí)別和融合至關(guān)重要[68]。當(dāng)CD9與特異性糖蛋白(Pregnancy-specific glycoprotein 17,PSG17)結(jié)合后，PSG17占據(jù)了配體融合結(jié)合位點(diǎn)，因而導(dǎo)致精卵不能發(fā)生融合[69]。

總之，只有精卵發(fā)生膜融合后，精子才得以穿入卵內(nèi)并逐漸形成雄原核，此時(shí)卵子受到激活并完成第二次減數(shù)分裂。隨著第二極體的排出，雌原核形成以及隨后兩性原核發(fā)生結(jié)合并形成受精卵。隨后受精卵不斷發(fā)育和成長(zhǎng)，受精卵開始第一次卵裂并完成精卵受精過(guò)程。受精卵在此后也依次經(jīng)歷4-細(xì)胞期、8-細(xì)胞期、16-細(xì)胞期、桑葚胚期、囊胚期等過(guò)程，最終形成胚胎乃至動(dòng)物個(gè)體。

表4 精子識(shí)別并融合卵膜的相關(guān)蛋白質(zhì)

圖5 精卵膜融合的過(guò)程

6 結(jié) 語(yǔ)

哺乳動(dòng)物受精作用受到多種蛋白分子調(diào)控，目前大部分蛋白分子在精子運(yùn)動(dòng)活力、精子獲能、頂體反應(yīng)以及精卵融合等方面的詳細(xì)作用機(jī)理仍不清楚。近年來(lái)，相關(guān)研究顯示，精子功能相關(guān)的蛋白質(zhì)能夠作為受精能力的標(biāo)記，評(píng)估或改善家畜的繁殖力。人工授精是應(yīng)用在畜牧業(yè)生產(chǎn)上最成功和廣泛的一項(xiàng)遺傳育種技術(shù)，但是如何客觀地評(píng)估和判斷精子受精能力是人工授精技術(shù)的關(guān)鍵之一。目前，畜牧生產(chǎn)上常用的評(píng)估方法是檢測(cè)精子的活力、密度以及形態(tài)等，然而這些傳統(tǒng)的評(píng)估方法并不能準(zhǔn)確判斷出公畜個(gè)體間受精能力的差異[70,71]。個(gè)體間受精能力的差異是由于精液中受精相關(guān)的蛋白分子組成不同所導(dǎo)致[72]，檢測(cè)出這些與受精能力相關(guān)的分子標(biāo)記或許是最為行之有效的評(píng)估方法。建立生物標(biāo)記檢測(cè)方法，人們可以有效地預(yù)測(cè)和評(píng)定公畜的繁殖力和受精性能，客觀地區(qū)分高低繁殖力的個(gè)體以獲得更好的生產(chǎn)效率。

雖然已證實(shí)一些蛋白分子(如 BSP、OPN、Acrosin、ADAMs和 CRISP1等)與精子功能以及公畜受精能力相關(guān)，但限于其實(shí)驗(yàn)的重復(fù)性和實(shí)際生產(chǎn)的應(yīng)用性，這些受精生物標(biāo)記還不足以客觀地應(yīng)用于評(píng)估個(gè)體的繁殖力。對(duì)精子發(fā)生、精子成熟、精子獲能以及頂體反應(yīng)等一系列受精過(guò)程需要進(jìn)行更深入地研究和探討，以尋找出合適于畜牧生產(chǎn)的生物標(biāo)記分子，進(jìn)而建立起更加簡(jiǎn)便、客觀和高效的受精能力評(píng)定方法。

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